(1) Field of the Invention
The present invention relates to segments of the Marek's Disease Herpesvirus genome, from its unique short (U.sub.S) region encoding glycoproteins gD, gI and gE, and to novel glycoproteins produced therefrom. In particular, the present invention relates to DNA segments containing genes encoding these glycoprotein antigens and containing potential promoter sequences up to 400 nucleotides 5' of each gene, segments which are useful for probing for Marek's disease herpesvirus, as a possible source for Marek's disease virus (MDV) promoters, for gene expression to produce the glycoproteins that in turn can be used for producing antibodies which recognize the three glycoprotein antigens, and in the case of the latter two genes, for potential insertion sites for foreign genes and as possible sites for gene inactivation to attenuate MDV field isolates for vaccine purposes.
(2) Prior Art
MDV is an oncogenic herpesvirus of chickens, which is known to cause T cell lymphomas and peripheral nerve demyelination. The resulting disease, Marek's disease (MD), was the first naturally occurring lymphomatous disorder to be effectively controlled via vaccination, using either the antigenically related, yet apathogenic, herpesvirus of turkeys (HVT) or attenuated field isolates of MDV.
Because of similar biological properties, especially its lymphotropism, MDV has been classified as a member of the gammaherpesvirus subfamily (Roizman, B., et al., Intervirology 16:201-217 (1981)). Of the three herpesvirus subfamilies, gammaherpesviruses exhibit particularly marked differences with regard to genome composition and organization. For example, the B-lymphotropic Epstein-Barr virus (EBV) of humans has a 172.3 kbp genome with 60% G+C content, is bounded by terminal 0.5 kbp direct repeats and contains a characteristic set of internal 3.07 kbp tandem repeats (Baer, R., et al., Nature (London) 310:207-211 (1984)). Herpesvirus saimiri (HVS), a T-lymphotropic herpesvirus of new-world monkeys and lower vertebrates, has an A+T rich coding sequence (112 kbp; 36% G+C; i.e. L-DNA) without any large-scale internal redundancy, but contains instead greater than 30 reiterations of a 1.44 kbp sequence of 71% G+C at the termini of the genome (H-DNA) (Banker, A. T., et al., J. Virol. 55:133-139 (1985)). Despite the structural differences between EBV and HVS, the genomes of these two viruses encode serologically related proteins and share a common organization of coding sequences which differs from that of the neurotropic alphaherpesviruses, exemplified by herpes simplex virus (HSV) and varicella-zoster virus (VZV) (Camerion, K. R., et al., J. Virol. 61:2063-2070 (1987); Davison, A. J., et al., J. Gen. Virol. 68:1067-1079 (1987); Davison, A. J., et al., J. Gen. Virol. 67:597-611 (1986); Davison, A. J., et al., J. Gen. Virol. 76:1759-1816 (1986); Davison, A. J., et al., J. Gen. Virol. 64:1927-1942 (1983); Gompels, U. A., J. of Virol. 62:757-767 (1988); and Nichols, J., et al., J. of Virol. 62:3250-3257 (1988)).
In contrast to other gammaherpesviruses, MDV has a genome structure closely resembling that of the alphaherpesviruses (Cebrian, J., et al., Proc. Natl. Acad. Sci. USA 79:555-558 (1982); and Fukuchi, K., et al., J. Virol. 51:102-109 (1984)). Members of the latter subfamily have similar genome structures consisting of covalently joined long (L) and short (S) segments. Each segment comprises a unique (U) segment (U.sub.L, U.sub.S) flanked by a pair (terminal and internals) of inverted repeat regions (TR.sub.L, IR.sub.L ; TR.sub.S ; respectively). Alphaherpesviruses include human HSV and VZV, porcine pseudorabies virus (PRV), bovine herpesvirus (BHV) and equine herpesvirus (EHV). Because MDV contains extensive repeat sequences flanking its U.sub.L region, its genome structure most resembles that of HSV (Cebrian, J., et al., Proc. Natl. Acad. Sci. USA 79:555-558 (1982); and Fukuchi, K., et al., J. Virol. 51:102-109 (1984)).
Recent studies (Buckmaster, A. E., et al., J. Gen. Virol. 69:2033-2042 (1988)) have shown that the two gammaherpesviruses, MDV and HVT, appear to bear greater similarity to the alphaherpesviruses, VZV and HSV, than to the gammaherpesvirus, EBV. This was based on a comparison of numerous randomly isolated MDV and HVT clones at the predicted amino acid level; not only did individual sequences exhibit greater relatedness to alphaherpesvirus genes than to gammaherpesvirus genes, but the two viral genomes were found to be generally collinear with VZV, at least with respect to the unique long (U.sub.L) region. Such collinearity of U.sub.L genes extends to other alphaherpesviruses such as HSV-1, HSV-2, EHV-1 and PRV as evidenced by both sequence analysis (McGeoch, D. J., et al., J. Gen. Virol. 69:1531-1574 (1988)) and DNA hybridization experiments (Davison, A. J., et al., J. Gen. Virol. 64:1927-1942 (1983)). Many of these U.sub.L genes are shared by other herpesviruses, including the beta- and gammaherpesviruses (Davison, A. J., et al., J. Gen. Virol. 68:1067-1079 (1987)). The organization and comparison of such genes has suggested the past occurrence of large-scale rearrangements to account for the divergence of herpesviruses from a common ancestor. Unfortunately, such a hypothesis fails to account for the presence of alphaherpesvirus S component (unique short, U.sub.S, and associated inverted/terminal repeat short, IR.sub.S, TR.sub.S) genes which appear unique to members of this subfamily (Davison, A. J., et al., J. Gen. Virol. 68:1067-1079 (1987); Davison, A. J., et al., J. Gen. Virol. 67:597-611 (1986); and McGeoch, D. J., et al., J. Mol. Biol. 181:1-13 (1985)).
The DNA sequence and organization of genes in a 5.5 kbp EcoRl fragment mapping in the U.sub.S region of MDV strain RBIB was described by Ross, Binns and Pastorek (Ross, L. J. N., et al, Journal of General Virology 72:949-954 (1991)). The properties and evolutionary relationships of four of the predicted polypeptides was also described (Ross, L. J. N. and M. M. Binns, Journal of General Virology, 72:939-947 (1991)). In that fragment they found the homologs of HSV US2, US3, US6 (gD) and US7 (gI), as well as an MDV specific gene. For the latter, only part of the gene was present. These reports confirm the presence of four MDV U.sub.S genes, and the evolutionary relationship proposed above. It is important to note that no evidence for US8 (gE), or the genes to the left of US2 were described.
In addition to its uniqueness compared with beta- and gammaherpesviruses, the alphaherpesvirus U.sub.S region is particularly interesting because of marked differences in its content and genetic organization within the latter subfamily (e.g. HSV-1 U.sub.S =13.0 kbp, 12 genes, McGeoch, D. J., et al., J. Mol. Biol. 181:1-13 (1985)); VZV U.sub.S =5.2 kbp, 4 genes, Davison, A. J., et al., J. Gen. Virol. 76:1759-1816 (1986)). In the case of HSV-1, 11 of the 12 U.sub.S genes have been found to be dispensable for replication in cell culture (Longnecker, R., et al., Proc. Natl. Acad. Sci. USA 84:4303-4307 (1987)). This has suggested the potential involvement of these genes in pathogenesis and/or latency (Longnecker, R., et al., Proc. Natl. Acad. Sci. USA 84:4303-4307 (1987); Meignier, B., et al., Virology 162:251-254 (1988); and Weber, P. C., et al., Science 236-576-579 (1987)). In the report by Buckmaster et al. (Buckmaster, A. E., et al., J. Gen. Virol. 69:2033-2042 (1988)), except for the identification of partial MDV sequences homologous to HSV immediate early protein alpha 22 (US1) and the serine-threonine protein kinase (US3), the content, localization and organization of MDV S component homologs was not determined. Moreover, despite the presence of at least four HSV U.sub.S glycoprotein genes (two in VZV), no such homologs were identified.
In application Ser. No. 07/229,011 filed Aug. 5, 1988, (now abandoned) including Leland F. Velicer, one of the present inventors, the Marek's Disease herpesvirus DNA segment possibly containing the gene encoding the glycoprotein B antigen complex (gp100, gp60, gp49) was identified but not sequenced. Antigen B is an important glycoprotein complex because it can elicit at least partial protective immunity, and thus MDV DNA segment can be used for probes, as a possible source for promoters in the gene's 5' regulatory region, and for gene expression to produce the glycoproteins, which in turn can be used to produce antibodies that recognize the glycoprotein antigens. However, there was no discussion of the glycoproteins of the present invention. These B antigen glycoproteins are not encoded by the U.sub.S region and thus are from a different region of the MDV genome.
In application Ser. No. 07/526,790, filed May 17, 1987 (now abandoned) by Leland F. Velicer, the MDV herpesvirus DNA segment containing the gene encoding the glycoprotein A antigen (gp57-65) is described but not sequenced. This MDV DNA segment is useful as probes, as a possible source for promoters in the gene's 5' regulatory region, and for producing antibodies by the sequence of events described above. This DNA is also important because antigen A is now known to be a homolog of HSV gC, a gene non-essential for replication in cell culture. Since that property most likely also applies to the MDV homolog, it may be useful as a site for insertion of foreign genes. However, there was no discussion of the glycoproteins of the present invention. This glycoprotein is also not encoded by the U.sub.S region and is thus from a different region of the MDV genome.
Other glycoproteins are encoded by Marek's disease herpesvirus genome. In application Ser. No. 07/572,711, filed Aug. 24, 1990 (now U.S. Pat. No. 5,138,033) by Leland F. Velicer, et al., the MDV DNA containing the genes encoding the MDV, gD, gI and part of gE glycoproteins is described, with MDV nucleotide sequences for the complete gD and gI genes and part of gE (MDV homologs of HSV genes US6, US7, US8, respectively). This MDV DNA segment is useful as probes, as a possible source for promoters in the gene's 5' regulatory region, and for producing antibodies by the sequence of events described above. The present invention is particularly directed to the complete gene (US8) encoding glycoprotein gE.